Our studies employing co-immunoprecipitation assays for dopamine receptors (DARs) coupled with mass spectrometry-based sequencing have identified sorting nexin-25 as a member of the DAR signalplex. Mammalian sorting nexins (SNXs) have been suggested to regulate intracellular trafficking, internalization, and endosomal recycling or sorting of membrane-bound cargo. The physiological role of SNX25 is unknown. Using RT-PCR we have found that SNX25 is expressed in multiple tissues including brain and kidney and that the endogenous isoform is longer than the one indicated in GenBank. The full-length SNX25 contains two putative transmembrane-spanning regions and confocal microscopy shows that it is localized in distinct clusters at or near the plasma membrane. When SNX25 is over-expressed with DARs, the receptors show changes in expression patterns and appear localized to the SNX25 clusters. Over-expression of SNX25 perturbed both endocytosis and recycling of the D2 DAR. Radioligand binding also show that the expression levels of both D1 and D2 DARs are increased with SNX25 over-expression. Decreasing the levels of endogenous SNX25 using siRNA causes a subsequent decrease in DAR expression. These data suggest that SNX25 plays a role in DAR trafficking through intracellular membrane compartments and regulates both receptor expression and signaling. We have also found that membrane lipid rafts are required for D2 receptor signaling. Lipid rafts are specialized membrane microdomains enriched in cholesterol, sphingolipids and caveolin and are important in the organization of GPCR-protein complexes and the regulation of signaling. Given the emerging significance of lipids with respect to GPCR structure and activation, we have investigated the role of lipid rafts and membrane cholesterol on D2 dopamine receptor (DAR) localization and function. Sucrose density fractionation revealed that D2 DARs are enriched in lipid rafts from both striatum and transfected HEK293cells. Depletion of membrane cholesterol with methyl-&#946;-cyclodextrin (MCD) diminished cell-surface D2 DAR expression in HEK293T cells by approximately 75% without altering total receptor number. MCD treatment also completely abolished DA signaling at the D2 DAR. Importantly, when D2 DAR transfections were titrated to achieve surface expression values comparable to MCD-treatment, a DA-dependent decrease in cAMP was still observed. Diminished signaling of Gi/o-coupled muscarinic receptors was also observed following MCD-treatments. Notably, MCD-treatment did not inhibit Gs-linked D1 DAR function or forskolin-stimulated cAMP accumulation. These results suggest that lipid rafts and/or membrane cholesterol are critical for D2 DAR signaling.